The ability to monitor the progression of the HIV virus in patients is important in the study of HIV transmission, in predicting the onset and advancement of the AIDS disease, and evaluating the efficacy of treatments. Present methods available to the clinical laboratory for studying HIV pathogenesis and transmission rely on surrogate markers. Surrogate markers are biological indicators that tend to reflect, to varying extents, the gradual progression from symptomless HIV infection to full blown AIDS. Presently, the most commonly used markers are immune system cells known as CD4 cells, which are infected and destroyed by the HIV virus, and HIV p24 antigen. Other biological materials such as beta-2 microglobulin, neopterin, interferon, other cytokines and their receptors, and even early clinical symptoms are some of the alternative markers which are being investigated.
The use of markers to evaluate HIV progression has a number of limitations. No known marker consistently reflects disease progression in all individuals. For example, quantitation of serum HIV p24 antigen is insensitive, because up to 60% of patients may not have detectable levels of p24 even using the immune complex dissociation assay. Measurement of absolute CD4+ T cells in persons given anti-retroviral therapy is increasingly being viewed as an inaccurate indicator of disease progression. Furthermore, co-infection with human T-cell leukemia virus (HTLV), which is prevalent in certain areas, is likely to further impair the reliability of CD4+ T cells as a marker for the HIV virus.
In addition, by overemphasizing one preferred marker such as CD4, researchers risk overlooking an effective treatment if the treatment happens not to affect the marker chosen. Although a marker must have a biologically plausible connection to disease progression, the connection cannot effectively be utilized in treatment evaluation unless the marker also responds quickly to effective treatment. The use of combinations of markers may cure some of these deficiencies, but not without a corresponding increase in complexity and potential for error.
Determining the total amount of HIV virus present in a person's system at a given time, i.e., the "viral load," has been suggested as a better way of predicting the progress of infection. The present methods for assessing viral load, however, are unsatisfactory. HIV gene amplification by polymerase chain reaction (PCR) is sensitive, but PCR-amplified products are highly susceptible to cross-contamination, making this method impractical for general clinical use. Plasma viral culture methods are complicated and susceptible to contamination. Such methods quantitate overall viral production in the patient, but do not enumerate the number of cells infected.
An alternative to PCR amplification and plasma viral culture is the cell dilutional viral co-culture method. In the co-culture method, HIV-seronegative donor cells (peripheral blood mononuclear cells (PBMCs)) are first stimulated with phytohemagglutinin (PHA), and then co-cultured with dilutions of patient PBMCs, generally for at least 21 days. In addition to requiring donor PBMCs, the co-culture method is labor intensive, and requires a frequent exchange of culture medium (every 3 to 4 days), increasing the possibility of microbial contamination.
Various stimuli are known to induce expression of HIV, including antigens, mitogens, UV radiation, and phorbol esters. Phorbol myristate acetate has been shown to increase HIV production in chronically infected clone cell lines, as described, for example, in Folks, et al., Characterization of a Promonocyte Clone Chronically Infected with HIV and Inducible By 13-Phorbol-12-Myristate Acetate, J. Immunol., v. 140, pp. 1117-22 (1988); Kinter, et al., Direct and Cytokine-Mediated Activation of Protein Kinase C Induces Human Immunodeficiency Virus Expression in Chronically Infected Promonocytic Cells, J. Virol., v. 64, pp. 4306-12 (1990); and Poli, et al., Interferon-.alpha. But Not AZT Suppresses HIV Expression in Chronically Infected Cell Lines, Science, v. 244, pp. 575-77 (1988). Also, stimulation of patients' PBMCs with PHA in combination with phorbol 12-myristate 13-acetate has resulted in augmentation of p24 antigen level expression, as described in Tetali, et al., Human Immunodeficiency Virus Type I RNA Detection in Peripheral Blood Mononuclear Cell by Polymerase Chain Reaction: Enhanced Sensitivity after Mitogenic Stimulation, AIDS Res. Hu. Retrovir., v. 9, pp. 77-82 (1993). The combined use of PHA and PMA on human PBMCs was intended to induce sufficient vital production for detection by mRNA PCR. p24 antigen production was measured in a few of the samples, however, the results were not correlated with patients' stage of disease.
In view of the above-noted deficiencies and complexities of the prior methods for assessing viral load, the need remains for a straightforward, rapid technique for determining HIV viral load.